桂枝茯苓汤含药血清对子宫内膜癌细胞的影响

Effect of Guizhi Fuling Decoction-Containing Serum on Endometrial Cancer Cells

为探究桂枝茯苓汤含药血清是否通过调控3磷酸肌醇激酶(PI3K)、蛋白激酶B(AKT),参与调控子宫内膜癌细胞增殖、上皮间质转化(EMT)与凋亡,将72只SD大鼠分为健康组、桂枝茯苓汤高剂量组、桂枝茯苓汤中剂量组、桂枝茯苓汤低剂量组和奥沙利铂组,对各组大鼠灌胃相应剂量药物,从大鼠的腹主动脉取血制备含药血清,培养子宫内膜癌HEC-B细胞,分为对照组、含药血清高剂量组、含药血清中剂量组、含药血清低剂量组、奥沙利铂血清组、含药血清高剂量+通路激活剂组,向各组细胞培养基中加入相应血清及试剂.采用四甲基偶氮唑蓝(MTT)法检测各组细胞增殖情况,流式细胞术检测各组细胞凋亡情况,Western Blot法检测EMT相关蛋白E钙黏蛋白(E-cadherin)、细胞波形蛋白(Vimentin)、N钙黏蛋白(N-cadherin)表达水平及PI3K/AKT通路相关蛋白磷酸化水平.结果表明:与对照组相比,含药血清高剂量组、含药血清中剂量组、含药血清低剂量组的细胞生存率、Vimentin和N-cadherin蛋白表达量、PI3K和AKT磷酸化程度均显著降低(P<0.05),细胞凋亡率、E-cadherin蛋白表达量显著升高(P<0.05),且呈剂量依赖性;与含药血清高剂量组相比,含药血清高剂量+通路激活剂组的细胞生存率、Vimentin和N-cadherin蛋白表达量、PI3K和AKT磷酸化程度均显著升高(P<0.05),细胞凋亡率、E-cadherin蛋白表达量显著降低(P<0.05),奥沙利铂血清组各项指标无显著差异(P>0.05).说明桂枝茯苓汤通过调控PI3K/AKT通路,抑制相关蛋白磷酸化,阻碍细胞生长,加剧细胞凋亡,缓解EMT,从而缓解子宫肌瘤病情.

The purpose of this study was to investigate the effect of Guizhi Fuling Decoction-containing serum in regulation of endometrial cancer cell proliferation,epithelium-mesenchymal transformation(EMT)and apoptosis through the regulation of inositol 3-phosphate kinase(PI3K)and protein kinase B(AKT).Seventy-two SD rats were divided into healthy group,high-dose Guizhi Fuling Decoction group,middle-dose Guizhi Fuling Decoction group,low-dose Guizhi Fuling Decoction group and oxaliplatin group.Corresponding doses of drugs were given by gavage,and blood was drawn from the abdominal aorta to prepare drug-containing serum.Human endometrial cancer HEC-B cells were cultured and grouped into control group,high-dose drug-containing serum group,medium-dose drug-containing serum group,low-dose drug-containing serum group,oxaliplatin serum group,high-dose drug-containing serum+pathway activator group.Corresponding serum and reagents were added to the cell culture medium of each group.The proliferation of cells in each group was detected by tetramethylazolyl blue(MTT)method;cell apoptosis in each group was detected by flow cytometry;the expression levels of EMT-related proteins E-cadherin(E-cadherin),cellular vimentin(Vimentin),N-cadherin(N-cadherin)and the phosphorylation levels of PI3K/AKT pathway-related proteins were detected by Western Blot.Compared with the control group,the cell survival rate,Vimentin,N-cadherin protein expression,PI3K and AKT phosphorylation levels in the high-dose drug-containing serum group,the medium-dose drug-containing serum group,and the low-dose drug-containing serum group were obviously decreased(P<0.05);while the apoptosis rate and E-cadherin protein expression were obviously increased(P<0.05),in a dose-dependent manner.Compared with the high-dose drug-containing serum group,the cell survival rate,Vimentin,N-cadherin protein expression,PI3K and AKT phosphorylation levels in the high-dose drug-containing serum+pathway activator group were obviously increased(P<0.05),and the apoptosis rate and E-cadherin protein expression were obviously decreased(P<0.05),and there was no significant difference in each index between the oxaliplatin serum group(P>0.05).Guizhi Fuling Decoction regulates the PI3K/AKT pathway to inhibit the phosphorylation of related proteins,hinder cell growth,aggravate cell apoptosis,relieve EMT,and thus alleviate the condition of uterine fibroids.